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系統識別號 U0026-0812200910430243
論文名稱(中文) 1. 受Ha-ras調控之基因BNIP3之選殖、特性及功能性研究 2. 利用Real-Time PCR偵測膀胱癌、結腸癌檢體中之H-ras及K-ras基因氨基酸12之突變
論文名稱(英文) 1. Cloning, characterization and functional study of BNIP3, a Ha-ras up-regulated gene 2. Screening codon 12 mutation of H-ras and K-ras genes in bladder and colon cancers by Real-Time PCR
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 91
學期 2
出版年 92
研究生(中文) 陳炳宏
研究生(英文) Ping-Hong Chen
學號 s4690105
學位類別 碩士
語文別 英文
論文頁數 85頁
口試委員 指導教授-劉校生
口試委員-呂增宏
口試委員-賴明德
口試委員-戴明泓
口試委員-林以行
中文關鍵字 膀胱癌 
英文關鍵字 Ras  Real-Time PCR  BNIP3 
學科別分類
中文摘要 Ras蛋白質在細胞內扮演一個訊息傳遞者的角色,調控細胞的增生、轉型、死亡。經由Ras蛋白質所誘導的細胞死亡機制被認為是一種防止細胞癌化的方法。Ras蛋白質可以在粒腺體上與Bcl-2蛋白質互相作用而調控細胞的生死。BNIP3 (Bcl-2 and Nineteen kDa Interacting Protein-3)屬於Bcl-2家族的BH3-domain only蛋白質。已知其可以與Bcl-2蛋白質結合,並會誘使細胞凋亡。本研究中顯示Ras和BNIP3蛋白質的表現在乳癌細胞株MCF7-ras、膀胱癌細胞株UB37, UB40及老鼠纖維母細胞株Bcl2-3A中均呈正相關。將可持續或誘導表現之BNIP3質體送入293細胞內大量表現時,造成該細胞之死亡。進一步分析發現這些細胞粒腺體膜通透性增加,但並不造成cytochrome C的釋放,而這些細胞是否死於細胞凋亡,仍未確定。此外也發現這些細胞中Ras蛋白質在粒腺體會與BNIP3蛋白質相互作用。綜述之,Ras可與Bcl-2及BNIP3;Bcl-2可與BNIP3相互結合,而三者之相互關係仍未可知,此外Ras與BNIP3正相關之作用機轉也有待進一步釐清。無論如何,Ras, Bcl-2和BNIP3可能在粒腺體上相互作用而進一步調節細胞的生死,這些假說均有待進一步之探討。
不同ras基因的點突變會改變其致癌能力。利用螢光探針及定量聚合酶儀器研發出一套快速篩檢臨床檢體中的K-ras及H-ras第12氨基酸突變的方法。藉由此方法在38個大腸癌檢體中偵測到8個檢體有K-ras codon 12突變,9個有H-ras codon 12突變;25個膀胱癌檢體中有8個H-ras codon 12突變且無K-ras codon 12突變。綜述之,本研究提供一個有別於傳統SSCP之方法用來快速、準確篩選Ras氨基酸12之突變,對於Ras相關之癌症診斷將有莫大之幫助。
英文摘要 Ras transduces multiple signals to modulate gene expression for cell proliferation, transformation and death. The cell death induced by Ras may play a critical role in preventing neoplasia formation. Mitochondria Ras interacts with Bcl-2 and regulates the death of cells. BNIP3 (Bcl-2 and Nineteen kDa Interacting Protein-3) belongs the BH3 domain only protein of Bcl-2 family. BNIP3 can bind with Bcl-2 and induces the death of mammalian cells. Our data showed that the expression of Ras correlated with the expression of BNIP3 in breast cancer cell MCF7-ras, mouse fibroblast NIH/3T3 cells Bcl2-3A, and bladder cancer cells, UB37, and UB40. Expression vector of BNIP3 was constructed, and overexpression of BNIP3 gene in 293 cells resulted in cell death and increase of mitochondria membrane permeability. Further study revealed that BNIP3 caused cell death is without release of cytochrome C. Our data also demonstrated that Ras interacted with BNIP3 on mitochondria. In conclusion, whether BNIP3 overexpression can induce the apoptosis of bladder cancer cell and whether the interaction among Ras, BNIP3 and Bcl-2 on mitochondria is involved in the cell death triggered by Ras-related BNIP3 remain unclear.
Different mutation types of three major Ras proteins (H-Ras, K-Ras and N-Ras) may lead to diverse carcinogenesis. For rapid screen of amino acid 12 mutants of Ras in clinical samples, a method using fluorescent probes and Real-Time PCR was established. A total of 8 mutations of K-ras and 9 mutations of H-ras were detected in 38 colon cancer samples. In addition, a total of 8 mutations of H-ras and no mutation of K-ras were detected in 25 bladder cancer samples. Compare to traditional methodology (SSCP), here we demonstrated a rapid and more precise method to screen codon 12 Ras mutations.
論文目次 I. Abstract
1. Abstract 1
2. 中文摘要 3
II. 誌謝 5
III. Contents 6
IV. Table list 8
V. Figure list 9
VI. Introduction 11
VII. Materials and methods
1. Bacterial and cell lines 16
2. Culture medium and condition 16
3. BNIP3 cDNA preparation 19
4. Plasmid and preparation 20
5. Screen and establish a stable cell
line harboring exogenous gene 22
6. Western blotting 24
7. Cell growth analysis 26
8. Viability stain 27
9. Immunoprecipitation 28
10. Cell cycle analysis 28
11. Mitochondria permeability assay 29
12. Apoptosis assay 30
13. RNA extraction 30
14. cDNA preparation 31
15. Gene expression quantification 31
16. K-ras codon 12 SNP determination 32
17. H-ras codon 12 SNP determination 34
VIII. Results
1. Relationship between Ras and BNIP3 36
2. Endogenous expression levels of
Ras in bladder cancer cell lines 37
3. Endogenous expression levels of BNIP3
gene in bladder cancer cell lines 38
4. Establish a stable cell line expressing
the inducible Ha-ras oncogene 38
5. Characterization of UB09 3-4 cells 38
6. Cloning of BNIP3 gene 40
7. K-ras codon 12 mutation 42
8. H-ras codon 12 mutation 44
IX. Discussion 46
X. References 51
XI. 自述 85
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